Thermocycling system and manufacturing method

ABSTRACT

A system for thermocycling biological samples within detection chambers comprising: a set of heater-sensor dies, each heater-sensor die comprising a heating surface configured to interface with a detection chamber and a second surface, inferior to the heating surface, including a first connection point; an electronics substrate, comprising a first substrate surface coupled to the second surface of each heater-sensor die, an aperture providing access through the electronics substrate to at least one heater-sensor die, and a second substrate surface inferior to the first substrate surface, wherein the electronics substrate comprises a set of substrate connection points at least at one of the first substrate surface, an aperture surface defined within the aperture, and the second substrate surface, and wherein the electronics substrate is configured to couple heating elements and sensing elements of the set of heater-sensor dies to a controller; and a set of wire bonds, including a wire bond coupled between the first connection point of at least one of the set of heater-sensor dies and one of the set of substrate connection points.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/879,517 filed 18-Sep.-2013, which is incorporated in its entiretyherein by this reference. This application is also related to U.S.application Ser. No. 14/487,808 filed 16-Sep.-2014, which isincorporated herein in its entirety by this reference.

TECHNICAL FIELD

This invention relates generally to the molecular diagnostics field, andmore specifically to an improved sample thermocycling system andassembly method thereof.

BACKGROUND

Molecular diagnostics is a clinical laboratory discipline that hasdeveloped rapidly during the last 25 years. It originated from basicbiochemistry and molecular biology research procedures, but now hasbecome an independent discipline focused on routine analysis of nucleicacids (NA), including deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) for diagnostic use in healthcare and other fields involvinganalysis of nucleic acids. Molecular diagnostic analysis of biologicalsamples can include the detection of one or more nucleic acid materialspresent in the specimen. The particular analysis performed may bequalitative and/or quantitative. Methods of analysis typically involveisolation, purification, and amplification of nucleic acid materials,and polymerase chain reaction (PCR) is a common technique used toamplify nucleic acids. Often, a nucleic acid sample to be analyzed isobtained in insufficient quantity, quality, and/or purity, hindering arobust implementation of a diagnostic technique. Current sampleprocessing methods and molecular diagnostic techniques are oftenlabor/time intensive, low throughput, and expensive, and systems ofanalysis are insufficient.

A rapid and efficient thermocycling system that can reliably thermocyclereagents used for processing of nucleic acids can significantly improvethe efficiency and effective implementation of molecular diagnostictechniques, such as realtime polymerase chain reaction (RT-PCR).Microfabrication techniques can produce such thermocycling systemscomprising precision heaters with low thermal masses and withwell-coupled temperature sensors. However, challenges are inherent inensuring that the microfabrication and assembly processes utilized tofabricate thermal cycling elements are extremely robust and reliable.

Due to these challenges and deficiencies of current molecular diagnosticsystems and methods, there is thus a need for an improved samplethermocycling system and assembly method thereof. This inventionprovides such a system and assembly method.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict embodiments of a thermocycling system;

FIG. 2 depicts an example schematic of a heater-sensor die duringfabrication in an example of a thermocycling system;

FIG. 3 depicts an example of heating and sensing elements in an exampleof a thermocycling system;

FIG. 4 depicts examples of additional elements of an embodiment of athermocycling system;

FIGS. 5A and 5B depict examples of reverse wire bonding in embodimentsof a thermocycling system;

FIGS. 6A-6D depict examples of reverse wire bonding in embodiments of athermocycling system;

FIGS. 7A-7B depict variations of configurations of elements in anembodiment of a thermocycling system;

FIGS. 8A-8C depict variations of configurations of elements in anembodiment of a thermocycling system;

FIGS. 9A-9C depict variations of configurations of additional elementsin an embodiment of a thermocycling system;

FIG. 10 depicts a flowchart of a method for assembling an embodiment ofa thermocycling system; and

FIGS. 11A-11B depict variations of a method for assembling an embodimentof a thermocycling system.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. System

As shown in FIGS. 1A and 1B, an embodiment of a sample thermocyclingsystem 100 comprises: a set of heater-sensor dies 110; an electronicssubstrate 140 configured to couple heating elements and sensing elementsof the set of heater-sensor dies to a controller; a set of heat sinksupports 150 coupled to at least one of the electronics substrate andthe set of heater-sensor dies; and a set of elastic elements 160 coupledto the electronics substrate and configured to bias each of the set ofheater-sensor dies against a detection chamber. In some embodiments, thesystem 100 further comprises a controller 165 coupled to the electronicssubstrate and configured to automate and/or control relevant heatingparameters of the system 100, and can further comprise a coolingsubsystem 170 configured to dissipate heat from the system 100. Thesystem 100 functions to enable rapid thermocycling of samples whileproviding uniform heating and preventing mechanical failure of thesystem 100 during thermocycling. In specific applications, the system100 can be used to rapidly and controllably thermocycle nucleic acidsamples during performance of molecular diagnostic amplificationtechniques (e.g., PCR, RT-PCR), signal amplification techniques (e.g.,bDNA, hybrid capture), and analytical techniques (e.g., gelelectrophoresis, mass spectrometry). In some variations, the system 100can be integrated into a molecular diagnostic system, such as thatdescribed in U.S. Pub. No. 2013/0210015, entitled “System and Method forProcessing and Detecting Nucleic Acids”, and filed on 13 Feb. 2013;however, the system 100 can additionally or alternatively be used withany other suitable system for processing biological or non-biologicalsamples.

1.1 Heater-Sensor Dies

The set of heater-sensor dies no functions to controllably heatindividual sample volumes. Preferably, each heater-sensor die 111 is athin-film die that can be deposited onto another substrate (e.g.,silicon substrate, glass substrate) that can be packaged onto anelectronics substrate 140; however, each heater-sensor die 111 canalternatively comprise any suitable geometry and/or configuration thatenables controlled, uniform, and rapid sample heating of a detectionchamber in thermal communication with the heater-sensor die 111. In someembodiments, the detection chambers can be those described in U.S. Pub.No. 2013/0209326, entitled “Microfluidic Cartridge for Processing andDetecting Nucleic Acids” and filed on 13 Feb. 2013, which is hereinincorporated in its entirety by this reference; however, the detectionchambers can alternatively be any other suitable container forprocessing a biological sample. Preferably, each heater-sensor die 111is characterized by a small profile (e.g., <10 mm dimension), whichensures that the heater-sensor die 111 is able to thermocycle extremelyrapidly; however, a heater-sensor dies 111 can alternatively becharacterized by any suitable profile. Additionally, each heater-sensordie 111 is preferably configured to conform to a detection chamber(e.g., sample tube, sample container, sample heating zone of a cartridgefor processing samples) configured to contain a sample during heating;however, a heater-sensor die 111 in the set of heater-sensor dies 110can alternatively not conform to a sample container. In one variation,each heater-sensor die 111 can be coupled to a thermally conductiveelement (e.g., 600 micron×5×5 mm silicon spacer) using thermallyconductive grease or another suitable material. In this variation, aconnection between a heater-sensor die is thus protected against failuredue to shear forces that can result from placement of a sample containeron a heater-sensor die 111. Other variations of preventing connectionfailure are described in Section 1.2 below.

Preferably, each heater-sensor die 111 in the set of heater sensor diesno comprises an insulating layer 112 that functions to provide aninsulating barrier to isolate the heaters and sensors and a heatingregion 113 that functions to provide uniform sample heating, as shown inFIG. 2. The insulating layer 112 is preferably electrically insulating,but can additionally be thermally insulating. Furthermore, eachheater-sensor die 111 preferably comprises two insulating layers 112that are configured to “sandwich” the heating region 113, thus isolatingthe heating region 113; however, each heater-sensor die 111 canalternatively comprise any suitable number of insulating layers 112arranged relative to the heating region 113 in any suitable manner. Theheating region 113 preferably comprises a heating element 114 with anintegrated sensing element 115, and is composed of a metal or metalalloy. Furthermore, the heating region 113 is preferably defined by apattern defined by geometric features (e.g., width, thickness, length,spacing) that facilitate uniform heating. However, in variations, theheating region 113 can alternatively not comprise an integrated sensingelement 115, can comprise any suitable number of heating elements114/sensing elements 115, and/or can be composed of any other suitablematerial.

In a first specific example of a heater-sensor die 111, as shown in FIG.3, a heater-sensor die 111 is configured to uniformly heat a circularregion having a diameter of 5 mm, spans a region of ˜8.6 mm×7 mm, andcomprises three heating elements 114 a, 114 b, 114 c: a central circularheating element 114 a and two circumferential heating elements 114 b,114 c configured to form a boundary about the central circular heatingelement. The heater-sensor die 111 in the first specific example furthercomprises three integrated sensing elements 115 a, 115 b, 115 c (i.e.,resistance temperature sensors, RTDs) distributed at three locationswithin the 5 mm circular region. In the first specific example, theheating region was 113 etched away in a boustrophedonic pattern,designed using a layout editor (e.g., Mentor Graphics™ or L-Edit™), toform the heating surface. The heating elements 114 are defined bycoarser patterning, and the sensing elements 115 are defined by finerpatterning, as shown in FIG. 3A. Other variations and examples of theheater-sensor dies 111 can comprise any suitable patterningconfiguration and/or any suitable arrangement of insulating layer(s) 112a, 112 b and heating region(s) 113.

1.2 Other System Elements

As shown in FIGS. 1A and 1B, the system 100 further comprises anelectronics substrate 140 and a set of heat-sink supports 150.Furthermore, the system 100 can additionally comprise a controller 165configured to automate and/or control relevant heating parameters of thesystem 100, and/or a cooling subsystem 170 configured to dissipate heatfrom the system 100, as shown in FIGS. 9A and 9B.

The electronics substrate 140 is preferably coupled to the set ofheater-sensor dies 110, and functions to enable communication betweeneach heater-sensor die 111 in the set of heater sensor dies 110 and acontroller 165. The electronics substrate 140 preferably comprises aprinted circuit board (PCB), and in some variations, comprises aflexible PCB, as shown in FIG. 4, in order to facilitate contact betweenheater-sensor dies 111 in the set of heater-sensor dies 110 anddetection chambers (e.g., reaction vessels, detection chambers) forprocessing according to molecular diagnostic protocols. Alternatively,the electronics substrate 140 can alternatively comprise a rigid PCB orany other suitable PCB. Furthermore, the system 100 can comprise anysuitable number of PCBs. Preferably, the set of heater-sensor dies 110is assembled onto the electronics substrate in a manner that providesthermal and/or electrical isolation of each die in from the neighboringdie(s), in particular, for variations wherein the electronics substrate140 is characterized as having poor conductivity. However, theelectronics substrate 140 and the set of heater-sensor dies no can beconfigured in any alternative suitable manner that provides isolation ofeach die in.

Preferably, the electronics substrate 140 is configured to couple to aheater-sensor die 111 by a reverse wire bond 145 coupled between a firstconnection point 146 (i.e., contact pad) of a set of connection points46 on the heater-sensor die 111 and a second connection point 147 (i.e.,pad) on the electronics substrate 140, as shown in FIGS. 4 and 5A-5B.The reverse wire bond 145 functions to prevent unbonding of aheater-sensor die 111 from the electronics substrate 140 that can resultfrom shear forces on at least one of the heater-sensor die 111 and thewire bond 145 and/or fatigue of the wire bond 145 during thermocycling.The reverse wire bond 145 can be made from a back-side of theelectronics substrate 140, in the orientation shown in FIGS. 5A-5B,through an aperture 143 defined within the electronics substrate 140.The aperture 143 can be a single aperture, or a set of aperturescorresponding to the set of heater-sensor dies 110, and furthermore,multiple heater-sensor dies 111 of the set of heater-sensor dies 110 canbe configured to couple to the electronics substrate 140 through anaperture 143 defined within the electronics substrate 140. As such, themappings between pads on the heater-sensor dies no and the electronicssubstrate 140 can be one-to-one or many-to-one in variations ofcoupling. In one variation, a set of apertures can be longitudinallyspaced across the electronics substrate 140; however, in othervariations, the set of apertures can be distributed across theelectronics substrate 140 in any other suitable manner. Also shown inFIGS. 5A-5B, coupling between the electronics substrate 140 and aheater-sensor die 111 can additionally comprise an adhesive layer 148comprising cyanoacrylate and/or any other suitable adhesive materialconfigured between the electronics substrate 140 and the heater-sensordie 111. In variations of heater-sensor die 111 coupling to theelectronics substrate 140 with an adhesive layer 148, the adhesive layeris preferably heat resistance in order to prevent failure at theadhesive layer 148 during thermocycling.

In a first variation, as shown in FIG. 6A, a heater-sensor die 111 isconfigured to couple to a first side 141 of the electronics substrate140 by a wire bond 145 that passes through an aperture 143 definedwithin the electronics substrate 140, such that the wire bond 145couples at one end to a second side 142 of the electronics substrate140. In this variation, a first connection point 146 on a surface of theheater-sensor die 111 closer to the first side 141 of the electronicssubstrate 140 is coupled to a second connection point 147 on the secondside of the electronics substrate 140, by way of the aperture 143 andthe reverse wire bond 145. In the first variation, the heater-sensor die111 can be further stabilized in place by an adhesive layer 148 locatedbetween the first side 141 of the electronics substrate 140 and theheater-sensor die 111. Furthermore, while one wire bond 145 isdescribed, the electronics substrate 140 can include a set of connectionpoints distributed at regions of the second substrate surface betweenadjacent apertures of a set of apertures of the electronics substrate140.

In a second variation, as shown in FIG. 6B, a heater-sensor die 111′ isconfigured to couple to a first side 141′ of the electronics substrate140′ by a wire bond 145′ that passes into an aperture 143′ definedwithin the electronics substrate 140. In this variation, the wire bond145 couples, at a first connection point 146′, to a surface of theheater-sensor die 111 closer to the first side 141′ of the electronicssubstrate 140′ and terminates at a second connection point 147′ part-waybetween a first side 141′ and a second side 142′ of the electronicssubstrate 140′, such that the wire bond 145′ is not exposed at thesecond side of the electronics substrate 140′. In the second variation,the heater-sensor die 111′ can also be stabilized in place by anadhesive layer 148′ located between the first side 141′ of theelectronics substrate 140′ and the heater-sensor die.

In a third variation, as shown in FIGS. 6C and 6D, a heater-sensor die111″ is configured to rest within a recess 149″ at the first side of theelectronics substrate 140″, wherein the recess 149″ is connected to(e.g., contiguous with) an aperture 143″ defined within the electronicssubstrate 140″. As shown in FIG. 6C, the recess 149″ can be configuredsuch that a heating surface of the heater-sensor die 111 is flush with afirst surface 141″ of the electronics substrate 140″; however, in anexample shown in FIG. 6D, the recess can also be configured such that aheating surface of the heater-sensor die 111″ is not flush with thefirst surface 141″ of the electronics substrate 140″. In the thirdvariation, the wire bond 145″ couples, at a first connection point 146″,to a surface of the heater-sensor die 111″ partially situated within theelectronics substrate 140″, and terminates at a second connection point147″ either part-way between the first side 141″ and a second side 142″of the electronics substrate 140″ (as in the second variation), or at asecond point 147″ at the second side 142″ of the electronics substrate140″ (as in the first variation). Thus, in the third variation, asurface of the heater-sensor die 111″ is stabilized within the recess149″ of the electronics substrate 140″ to further prevent shearing orother forms of mechanical failure that could compromise coupling betweenthe heater-sensor die 111″ and the electronics substrate 140″. In thethird variation, the heater-sensor die 111″ can also be furtherstabilized within the recess 149″ by an adhesive layer 148″ between therecess 149″ and the heater-sensor die 111″. While a single recess isdescribed, the electronics substrate 140″ can include a set of recess,each contiguous with at least one aperture of set of apertures of theelectronics substrate 140.

Other variations of the reverse wire bond(s) 145 between a heater-sensordie 111 and the electronics substrate 140 can comprise any suitablecombination of the above variations, and can additionally oralternatively comprise any suitable encapsulation, embedding, or pottingof wire bonds to further prevent failure in the wire bonds.

In still other variations, each heater-sensor die 111 can be coupled tothe electronics substrate 140 by any other suitable method. In onevariation, the coupling can comprise a “top-side” wire bond 145 b, asshown in FIG. 7A. In this variation, a thin wire (e.g., 10-300 micronsthick) composed of an electrically conductive material (e.g., aluminum,gold, or copper wire) is coupled between a first connection point 146 onthe heater-sensor die 111 and a second connection point 147 on a secondside 142 of the electronics substrate 140, in the orientation shown inFIG. 7A. Furthermore, in this variation, the “top-side” wire bonds 145 bare potted, embedded, and/or encapsulated to protect them frommechanical failure. In another variation, the coupling can comprise aflip-chip bond 145 c, as shown in FIG. 7B. In this variation, a volume(e.g., ball) of solder is placed between a first connection point 146 ona heater-sensor die 111 and a second connection point 147 on theelectronics substrate 140. Furthermore, in this variation, a fillermaterial 148 b can be placed in regions between the electronicssubstrate 140 and the heater-sensor die 111 not connected by a volume ofsolder of the flip-chip bond 145 c. In other variations, the couplingcan additionally or alternatively comprise any suitable adhesive (e.g.,cyanoacrylate adhesive) layer 148 configured between the heater-sensordie 111 and the electronics substrate 140.

The set of heat sink supports 150 is preferably coupled to at least oneof the set of heater-sensor dies no and the electronics substrate 140and functions to facilitate rapid thermocycling by dissipating heat fromthe set of heater-sensor dies 110 and/or the electronics substrate 140.The set of heat sink supports 150 can further function to providestructural support for the set of heater-sensor dies 110, such that theset of heater-sensor dies no is supported during compression (e.g.,compression against a set of detection chambers) and/or tension. In theabsence of heat sinking, the electronics substrate 140 and thesurrounding environment can potentially retain too much heat, whichcompromises the cooling of the set of heater-sensor dies 110. The set ofheat sink supports 150 can comprise multiple heat sink supports 151configured to define any suitable number of contact locations, or canalternatively comprise a single heat sink support 151 configured todefine any suitable number of contact locations. As shown in FIGS. 6Aand 6B, the system 100 preferably couples to a detection chamber (e.g.,reaction vessel, detection chamber) from a first side 101 a of thesystem, which can restrict heat dissipation from the first side 101 a ofthe system. Furthermore, the second side 101 b of the system 100 istypically used for optical imaging for monitoring (e.g., realtimemonitoring, delayed monitoring), and further limiting heat-sinking fromthe second side 101 b. Thus, it is preferable for the set of heat sinksupports 150 to couple to the system 100 from a side of the system 100that does not physically interfere with optical imaging apparatusinterfacing with the system 100. However, alternative configurations ofthe set of heat sink supports 150 can comprise coupling at any suitableside and/or any number of sides of the system 100.

As shown in FIGS. 8A and 8B, the set of heat sink supports 150 can beconfigured in any of a number of variations. In a first variation, eachheat sink support 151 can be directly placed against a first surface 105a of heater-sensor die 111 opposing that of a second surface 105 bcontacting a detection chamber, as shown in FIG. 7A. The first variationenables efficient transfer of heat out of the first surface 105 a of theheater-sensor die away from a respective detection chamber; however,excessive heat sinking can affect heating ramp rates. In a secondvariation, the system 100 comprises a thermally insulating assembly 152between a heater-sensor die 111 and a corresponding heat sink support151, as shown in FIG. 7B. In the second variation, the electronicssubstrate 140 can serve as the thermally insulating assembly 152 and canbe situated between the heater-sensor die 111 and a heat sink support151. Furthermore, in the second variation, a suitable thermal resistanceprovided by the electronics substrate 140 (e.g., through thickness,material selection, a combination of features) could produce a thermalcouple between the heater-sensor die 111 and the heat sink support 151to permit the heating capacity of the heater-sensor die 111 to achievethe heating times and/or heating ramp rate required by the application,while still allowing adequate cooling rates. Additionally, the secondvariation can provide increased backside support to each of the set ofheater-sensor dies 110 as well as increased surface for adhesion.

In specific examples of the second variation, heat sinking andsupporting the “backside” of the electronics substrate 140 can beimplemented across multiple heater-sensor dies in, separated by Societyfor Laboratory Automation and Screening (SLAS) standard spacings, suchas 9 mm, 4.5 mm or 2.25 mm spacings. The heat sink support 151 material(e.g., aluminum, copper, silver) in the specific examples is mated withthe electronics substrate 140 at each heater-sensor die location, withan air gap positioned laterally between each heater-sensor die location.This configuration can further function to reduce cross talk across aset of detection chambers in contact with the set of heater-sensor diesno. The set of heat sink supports 150 can, however, be configured in anyother suitable manner to provide heat dissipation within the system 100,without obstruction of optical detection apparatus, and with provisionof desired heat ramping and/or cycling behavior.

In specific examples of the second variation, heat sinking andsupporting the backside (i.e., first side 141) of the electronicssubstrate 140 can be implemented across multiple heater-sensor dies in,separated by Society for Laboratory Automation and Screening (SLAS)standard spacings, such as 9 mm, 4.5 mm or 2.25 mm spacings. The heatsink support 151 material (e.g., aluminum, copper, silver) in thespecific examples is mated with the electronics substrate 140 at eachheater-sensor die location, with an air gap between locations. Thisconfiguration can further function to reduce cross talk between at leasta first detection chamber and a second detection chamber interfacingwith the system 100.

The set of elastic elements 160 is preferably coupled to a first surface104 a of the electronics substrate 140, and functions to promote contactbetween the set of heater-sensor dies 110 and detection chambers (e.g.,reaction vessels, detection chambers) for sample processing according tomolecular diagnostic protocols. The set of elastic elements 160 cancomprise any one more of springs and elastomeric elements, which candeform and provide transmit a biasing force, through the electronicssubstrate 140, to reinforce contact between a set of detection chambersand the set of heater-sensor dies 110. The set of elastic elements 160can, however, additionally or alternatively include any other suitableelements configured to provide a biasing force that reinforces contactbetween a set of detection chambers and the set of heater-sensor dies110 in an elastic or a non-elastic manner. In one such alternativevariation, the system 100 can include one or more actuators configuredto drive each of the set of heater-sensor dies in toward a correspondingdetection chamber, and in another such alternative variation, the system100 can include a set of magnets (e.g., including magnet pairssurrounding the set of heater-sensor dies no and a corresponding set ofdetection chambers), that function to reinforce coupling between the setof heater-sensor dies 110 and the set of detection chambers. However,any other suitable elements can additionally or alternatively be used tofacilitate uniform and consistent coupling between the set ofheater-sensor dies 110 and a set of detection chambers.

In embodiments of the system 100 including a set of elastic elements160, the set of elastic elements 160 is preferably coupled to a firstsurface 104 a of the electronics substrate 140, as shown in FIG. 8C,such that each elastic element in the set of elastic elements 160facilitates contact between a heater-sensor die 111 and a correspondingdetection chamber. In a first variation, the set of elastic elements 160is coupled to first surface 104 a of a flexible PCB of the electronicssubstrate 140, as shown in FIG. 5A. In the first variation, contactbetween each heater-sensor die 111 and a corresponding detection chamberis thus maintained by a biasing force provided by an individual springthrough the flexible PCB of the electronics substrate 140. In the firstvariation, the number of elastic elements in the set of elastic elements160 is equal to the number of heater-sensor dies in the set ofheater-sensor dies 110, such that the set of elastic elements 160 andthe set of heater-sensor dies 110 are paired in a one-to-one manner.Alternatives to the first variation can, however, comprise any suitablenumber of elastic elements in relation to a number of heater-sensor dies110. In a second variation, the set of heater-sensor dies 110 is coupledto a second surface 104 b of a rigid PCB of the electronics substrate140, with the set of elastic elements 160 coupled to the first surface104 a of the rigid PCB. In the second variation, the set of elasticelements 160 thus functions to collectively transfer a force through therigid PCB to maintain contact between the set of heater-sensor dies 110and the detection chambers. Alternatives to the second variation canalso comprise any suitable number of springs in relation to a number ofheater-sensor dies in the set of heater-sensor dies 110. Furthermore,variations of the system 100 can include one or more elastic elementscoupled to any other elements directly or indirectly coupled to the setof heater-sensor dies 110. For instance, the system 100 can additionallyor alternatively include one or more springs 160′ coupled to basesurfaces of the set of heat-sink supports 150 interfacing with the setof heater-sensor dies, in order to transmit biasing forces.

As shown in FIG. 1, the system 100 can further comprise a controller165, which functions to automate and/or control heating parametersprovided by the set of heater-sensor dies no. The controller 165 canfurther be configured to provide heat parameter output commands to theheating element(s) 114, and/or configured to receive communication ofheating parameters (e.g., detected temperatures) sensed at the sensingelement(s) 115 of the system 100. The controller 165 preferablycomprises a proportion-integral-derivative (PID) controller, but canalternatively be any other suitable controller 165. The controller 165preferably interfaces with the set of heater-sensor dies no through theelectronics substrate 140 by a connector; however, the controller 165can interface with the set of heater-sensor dies no in any alternativesuitable manner. Preferably, the controller 165 is configured toautomate and control heat output parameters, including any one or moreof: heating temperatures, heating ramp rates, heating times (e.g.,holding times), and any other suitable heating parameter(s).Furthermore, the controller 165 can be configured to control individualheater-sensor dies 111 in order to provide unique heating parameters forindividual detection chambers and/or can be configured to provide commonheating parameters for all heater-sensor dies 111 in the set ofheater-sensor dies no. In a specific example, the controller 165comprises a Yokogawa UT750 PID controller, an Arduino UNO R3microcontroller configured to cycle the UT750 through temperature stagesand to control temperature holding, a resistance-to-voltage conversioncircuit, and two power supplies—a first power supply configured tosupply power to the set of heater-sensor dies 110 and a second powersupply configured to supply voltage to the resistance-to-voltageconversion circuit. In the specific example, the controller 165comprises a resistance-to-voltage conversion circuit because the UT750PID controller requires voltage as an input for PID control. In anotherspecific example, the controller 165 comprises a National InstrumentsLabView based system comprised of an NI cDAQ-9178 chassis with an NI9219 universal analog input card and an NI 9485 eight-channelsolid-state relay sourcing or sinking digital output module solid-staterelay card. In this specific example, the cDAQ-9178 supports the NI 9219and NI 9485 cards, the NI 9219 is used to obtain the RTD inputs, and theNI 9485 cycles the power supply voltage to individual heater-sensor diesof the set of heater-sensor dies 110. Further, in this specific example,the controller 165 is expandable to 12 or more channels through the useof additional NI 9219 and NI 9485 cards, each of which can handleseveral channels.

As shown in FIGS. 9A and 9B, the system 100 can further comprise acooling subsystem 170, which functions to provide heat transfer from thesystem 100 in order to further enhance controlled heating and cooling bythe system 100. The cooling subsystem 170 is preferably configured toprovide at least one of convective cooling and conductive cooling of thesystem 100, but can alternatively be configured to provide any othersuitable cooling mechanism or combination of cooling mechanisms. In onevariation, the cooling subsystem 170 can comprise a fan 171 thatprovides convective heat transfer from the system 100. In thisvariation, the fan 171 can be coupled to any suitable element of thesystem 100, such as the set of heat sink supports 150, as shown in FIG.9A. Furthermore, alternatives to this variation can comprise anysuitable number of fans of any suitable dimension and configuration,examples of which are shown in FIGS. 9A and 9B. In one such example, thesystem can include a set of cooling elements integrated with each heatsink support of the set of heat sink supports. In another variation, thecooling subsystem 170 can additionally or alternatively comprise aPeltier device, as shown in FIG. 9C. The Peltier device can be cooledand maintained at a defined temperature (e.g., in the 10-25 C range) toprovide a substantial temperature gradient for cooling during a thermalcycling process, which can decrease cooling times and/or cycle times. Inyet another variation, the cooling subsystem 170 can additionally oralternatively comprise a liquid cooling system (e.g., water coolingsystem) configured to surround and absorb heat from one or moreheater-sensor dies of the set of heater-sensor dies no, for instance, byway of the set of heat sink supports 150. The cooling subsystem 170 canadditionally or alternatively comprise any other suitable coolingelement(s).

In some variations, reflection from the set of heater-sensor dies no caninterfere with light transmitted to photodetectors of an opticalsubsystem opposed (e.g., directly opposed, in opposition) to the set ofheater-sensor dies no (e.g., light emitted from the set of biologicalsamples, light transmitted through filters of an optical subsystem),especially in configurations wherein a set of detection chambers isconfigured between the set of heater-sensor dies and optical elements ofan optical subsystem. In these variations, the set of heater-sensor dies110 can include elements that reduce or eliminate reflection from anyportion of the set of heater-sensor dies (e.g., reflection from theheating region, etc.), thereby facilitating analysis of a set ofbiological samples within the set of detection chambers. In onevariation, the set of heater-sensor dies 110 can include or be coupledto one or more non-reflective coatings 180 at surfaces of the set ofheater-sensor dies 110 upon which light from the optical subsystemimpinges. In a specific example, the non-reflective coating 180 cancomprise a high-temperature paint (e.g., dark paint, flat paint) thatfunctions to absorb and/or diffuse light from an opposing opticalsubsystem, while facilitating heat transfer to a set of detectionchambers in thermal communication with the set of heater-sensor dies no.In another variation, the set of heater-sensor dies no can be configuredto be in misalignment with photodetectors of the optical subsystem, suchthat reflection does not interfere with light transmitted to thephotodetectors of the optical subsystem. In one example, the set ofheater-sensor dies no can be configured to heat a set of detectionchambers from a first direction, and the optical subsystem can beconfigured to receive light from the set of detection chambers from asecond direction (e.g., a direction non-parallel to the firstdirection), such that reflection from the set of heater-sensor dies nodoes not cause interference. In still other variations, the set ofheater-sensor dies no can include any other suitable elements (e.g.,coatings, layers, etc.) and/or be configured in any other suitablemanner that eliminates, prevents, or mitigates reflection from the setof heater-sensor dies 110 from interfering with light transmitted tophotodetectors of an optical subsystem in opposition to the set ofheater-sensor dies 110.

Variations of the system 100 can, however, include any other suitableelement(s) configured to provide uniform, accurate, precise, andreliable heating of one or more detection chambers in thermalcommunication with the system 100. Furthermore, as a person skilled inthe art will recognize from the previous detailed description and fromthe figures, modifications and changes can be made to the preferredembodiments of the system 100 without departing from the scope of thesystem 100.

2. Method of Assembly

As shown in FIG. 10, a method 200 of assembling an embodiment of athermocycling system 100 comprises forming a first insulating layercoupled to exposed surfaces of a substrate S210, depositing a heatingregion upon the first insulating layer S220, producing a heating patternwithin the heating region, thus producing a heating array S230, dividingthe heating array into a set of heater-sensor dies S240, and couplingthe set of heater-sensor dies to a electronics substrate S250. Themethod 200 can further comprise forming a second insulating layer uponthe heating region S260, which functions to electrically isolate theheating region on a first side and a second side. The method 200functions to produce a thermocycling system 100, embodiments,variations, and examples of which are described above, wherein thethermocycling system 100 provides rapid and uniform thermocycling ofsamples and comprises elements configured to prevent mechanical failure.

Block S210 recites: forming a first insulating layer coupled to exposedsurfaces of a substrate, and functions to generate a layer thatelectrically insulates the heating region deposited in Step S220. Thesubstrate is preferably a silicon substrate, but can alternatively beany other suitable semi-conducting, or non-conducting substrate. Assuch, in variations, the substrate can be composed of a semi-conductingmaterial (e.g., silicon, quartz, gallium arsenide), and/or an insulatingmaterial (e.g., glass, ceramic). In some variations, the substrate 130can even comprise a combination of materials (e.g., as in a composite,as in an alloy). In examples wherein the substrate is a siliconsubstrate, the substrate can be composed of silicon with any suitabletype (e.g., P-type), doping (e.g., boron-doping), miller indexorientation, resistivity, thickness, total thickness variation, and/orpolish.

In forming the first insulating layer, Block S210 can be performed usingany one or more of: thermal oxide growth, chemical vapor deposition(CVD), spin coating, spray coating, and any other suitable method ofdepositing a localized layer of an insulating material. Preferably, thefirst insulating layer is composed of an insulating oxide material, andin examples can include any one or more of: a thermally grown siliconoxide, a chemical vapor deposited oxide, a deposited titanium oxide, adeposited tantalum oxide, and any other suitable oxide grown and/ordeposited in any other suitable manner. However, the first insulatinglayer can additionally or alternatively include an insulating polymer(e.g., a polyimide, a cyanate ester, a bismaleimide, a benzoxazine, aphthalonitrile, a phenolic, etc.) that is chemical and heat resistantand/or any other suitable material (e.g., chemical vapor depositednitride, other nitride, paralene, etc.) that is configured to providethe first insulating layer.

In one example of Block S210, the first insulating layer comprises anoxide material, and is formed by growing the oxide material on asubstrate. In one example of Block S210, the insulating layer comprisesa 0.2 mm layer of silicon oxide, and is formed on a 100 mm silicon waferusing thermal oxidation at 900° C. using water vapor (i.e., in wetoxidation) or oxygen (i.e., in dry oxidation) as the oxidant. Inalternative variations and examples of Block S210, the first insulatinglayer can be formed using high or low temperature thermal oxidation,using any suitable oxidant, and/or using any other suitable method(e.g., fluid deposition of an electrically insulating polymer,softbaking/hardbaking of a deposited polymer, etc.).

Block S220 recites depositing a heating region upon the insulatinglayer, and functions to form a thermally conductive substrate that isrobust during rapid thermocycling. Preferably, the heating regioncomprises a metal or a metal alloy and can comprise multiple layers;however, the heating region can alternatively comprise any suitablethermally conducting material, and can comprise any suitable number oflayers. Additionally, the heating region is preferably deposited in auniform layer; however, the heating region can be depositednon-uniformly in other variations. In one variation, the heating regioncomprises an adhesion material layer and a noble material layer, whereinthe noble material layer is deposited upon the adhesion material layerafter the adhesion material layer is deposited upon the first insulatinglayer. In examples of this variation, the adhesion layer can comprisechromium or titanium, and the noble layer can comprise gold or platinum.In one example of Block S220, the conductive material(s) of the heatingregion is(are) deposited using an evaporation process; however, in otherexamples, the conductive material(s) can be deposited by sputtering,plating (e.g., chemical plating, electrochemical plating), or any othersuitable method (e.g., electrodeposition). Furthermore, in exampleswherein a heating region material is evaporated or sputtered, theinsulating layer-substrate subassembly generated in Block S210 can betranslated or rotated in order to facilitate uniform deposition of theheating region material.

Block S230 recites producing a heating pattern within the heatingregion, and functions to produce a heating array characterized by aheating pattern that provides uniform heating and desirable resistancesfor heating elements and/or sensing elements (e.g., RTDs) defined withinthe heating region. As such, Block S240 preferably produces a heatingpattern having geometric features (e.g., width, thickness, length,spacing) that facilitate uniform heating and provide desired heating andsensing characteristics (e.g., resistance characteristics). In somevariations, the pattern can define any one or more of: linear segments,non-linear segments, boustrophedonic segments, continuous segments,non-continuous segments, and any other suitable segment(s) having anyother suitable geometry (i.e., width, depth, height, length, path, etc.)In a specific example, the heating pattern was designed using a layouteditor (e.g., Mentor Graphics™ or L-Edit™), and comprises aboustrophedonic pattern that is coarse for heating elements and fine forsensing elements. In alternative variations, the heating pattern can bedesigned using any other suitable method, and can alternatively oradditionally comprise any features that contribute to uniform heatingand/or suitable resistance ranges. During implementation of Block S230,the heating pattern can be produced photolithographically using apositive resist process, as shown in FIG. 11A. In one example, theheating region can be covered with positive photoresist (e.g., aphotomask designed according to the heating pattern) andlithographically etched in exposed regions. In the example, the positivephotoresist can then be removed to reveal the heating pattern. In othervariations, the heating pattern can be produced using any lithographicmethod, using positive and/or negative etching to form the heatingpattern, and/or using any other suitable method. In one example of analternative implementation of Step S230′, the heating pattern can beproduced using a lift-off process, as shown in FIG. 11B, wherein asacrificial layer is used to define the heating pattern, the heatingregion material(s) is(are) deposited, and then the sacrificial layer isremoved to reveal the heating pattern.

Block S240 recites dividing the heating array into a set ofheater-sensor dies, and functions to divide the heating array into a setof heater-sensor dies configured to heat multiple detection chambers(e.g., reaction vessels, sample containers, wells of a plate, chambersof a cartridge) in parallel (e.g., simultaneously, in sequence). BlockS240 can also comprise cleaning and drying the heating array prior toand/or after dividing the heating array into a set of heater-sensordies. Upon completion of Block S240, individual heater-sensor dies ofthe set of heater-sensor dies can be coupled to one or multipleelectronics substrates in order to provide uniform heating of individualsample containers with independent control of heating parametersprovided at each of the set of heater-sensor dies. In Block S240, theheating array is preferably divided using a dicing method (e.g.,mechanical dicing by saw, laser dicing, water cutting, stealth dicing,etc.), but can additionally or alternatively be divided using any othersuitable method (e.g., dice before grind). Furthermore, the heatingarray is preferably divided into rectangular dies, but can alternativelybe divided into dies of any suitable morphology (e.g., polygonal dies,non-polygonal dies, circular dies, ellipsoidal dies, etc.). In aspecific example, as shown in FIG. 3A, each heater-sensor die producedafter division of the heating array has dimensions of approximately 8.6mm×7 mm, with a circular heating surface that is 5 mm in diameter.

Block S250 recites coupling the set of heater-sensor dies to anelectronics substrate, and functions to provide a set of robustconnections between the set of heater-sensor dies and an electronicssubstrate. Coupling in Step S250 comprises forming an electricalconnection between connection points on the heater-sensor dies and theelectronics substrate, which enables driving of a heating current fromthe electronics substrate to each of the set of heater-sensor dies(e.g., simultaneously, non-simultaneously). The electrical connectioncan be provided by a conducting wire (e.g., aluminum wire, gold wire,copper wire) of any suitable thickness (e.g., 10-200 microns), or bysoldering. Furthermore, coupling in Block S250 preferably comprisescoupling heater-sensor dies with a suitable center-to-center spacingthat accommodates the spacing of detection chambers intended to beheated by the system. In a specific example, coupling in Block S250comprises providing a center-to-center spacing between heater-sensordies of 9 mm, 4.5 mm, or 2.25 mm according to Society of LaboratoryAutomation Standards (e.g., SLAS Microplate Standards).

Preferably, for a heater-sensor die, Block S250 comprises forming areverse wire bond between a connection point (i.e., pad) on theheater-sensor die and a connection point (i.e., pad) on the electronicssubstrate, as shown in FIGS. 5A-5B and 6A-6D. The reverse wire bondprevents unbonding of a heater-sensor die from the electronicssubstrate, which can be caused due to mechanical forces on the wire bondand/or heater-sensor die, or fatigue failure of a connection. In BlockS250, the reverse wire bond is preferably made from a back-side of theelectronics substrate, in the orientation shown in FIGS. 5A-5B, throughan aperture defined within the electronics substrate. The aperture canbe a single aperture, or a set of apertures, and furthermore, multipleheater-sensor dies of the set of heater-sensor dies 110 can beconfigured to couple to the electronics substrate through an aperturedefined within the electronics substrate. The mappings between pads onthe heater-sensor dies and the electronics substrate can be one-to-oneor many-to-one in variations of coupling. Also shown in FIGS. 5A-5B,Block S250 can additionally comprise depositing an adhesive layercomprising cyanoacrylate and/or any other suitable adhesive materialbetween a heater-sensor die and the electronics substrate.

In a first variation, as shown in FIG. 6A, Block S250 comprises couplinga heater-sensor die to a first side of the electronics substrate by awire bond that passes through an aperture defined within the electronicssubstrate, such that the wire bond couples at one end to a second sideof the electronics substrate. In this variation, a connection point on asurface of the heater-sensor die closer to the first side of theelectronics substrate is coupled to a connection point on the secondside of the electronics substrate, by way of the aperture and thereverse wire bond. In the first variation of Block S250, theheater-sensor die can be further stabilized in place by depositing anadhesive layer at the first side of the electronics substrate.

In a second variation, as shown in FIG. 6B, Block S250 comprisescoupling a heater-sensor die to a first side of the electronicssubstrate by a wire bond that passes into an aperture defined within theelectronics substrate. In this variation, the wire bond is configured tocouple, at one end, to a surface of the heater-sensor die closer to thefirst side of the electronics substrate, and configured to terminate ata connection point part-way between a first side and a second side ofthe electronics substrate, such that the wire bond is not exposed at thesecond side of the electronics substrate. In the second variation ofBlock S250, the heater-sensor die 111 can also be stabilized in place bydepositing an adhesive layer at the first side of the electronicssubstrate.

In a third variation, as shown in FIGS. 6C-6D, Block S250 comprisesproviding a recess within the electronics substrate at a first side ofthe electronics substrate, and coupling (e.g., mounting) a heater-sensordie within the recess of the electronics substrate, wherein the recessis connected to (e.g., contiguous with) an aperture defined within theelectronics substrate. Providing the recess of the third variationpreferably comprises forming the electronics substrate with a recess andaperture contiguous with the recess, wherein examples of forming caninclude any one or more of: molding (e.g., injection molding), casting,printing (e.g., 3D printing), and any other suitable method of formingthe electronics substrate. Providing the recess of the third variationcan additionally or alternatively comprise a method of removing materialfrom a substrate, such as etching, machining (e.g., drilling, milling),and any other suitable method of material removal.

As shown in FIG. 6C, a first example of providing the recess of BlockS250 can include providing a recess that is configured such that aheating surface of the heater-sensor die is flush with a first surfaceof the electronics substrate; however, in an example shown in FIG. 6D,providing the recess can include providing a recess that is configuredsuch that a heating surface of the heater-sensor die is not flush withthe first surface of the electronics substrate. In the third variationof Block S250, the wire bond is configured to couple, at one end, to asurface of the heater-sensor die partially situated within theelectronics substrate, and configured to terminate at a connection pointeither part-way between the first side and a second side of theelectronics substrate (as in the second variation of Block S250), or ata termination point at the second side of the electronics substrate (asin the first variation of Block S250). Thus, the third variation ofBlock S250 comprises stabilizing a surface of the heater-sensor diewithin the recess of the electronics substrate to further preventshearing or other forms of mechanical failure that could compromise aconnection between the heater-sensor die and the electronics substrate.In the third variation, the heater-sensor die can also be furtherstabilized by providing an adhesive layer within the recess.

In other variations, Block S250 can comprise coupling each heater-sensordie to the electronics substrate by any other suitable method. In onesuch variation of Block S250, coupling can comprise a “top-side” wirebond, in the orientation shown in FIG. 7A. In this variation, Block S250comprises coupling a thin wire (e.g., 10-300 microns thick) composed ofan electrically conductive material (e.g., aluminum, gold, or copperwire) between a connection point on the heater-sensor die and aconnection point on the top side electronics substrate (in theorientation shown in FIG. 7A). Furthermore, in this variation, the“top-side” wire bonds are potted, embedded, or encapsulated to protectthem from mechanical failure. In another alternative variation, couplingin Block S250 can comprise forming a flip-chip bond, as shown in FIG.7B. In this variation, Block S250 can include providing a volume (e.g.,ball) of solder configured between a connection point on a heater-sensordie and a connection point on the electronics substrate. Furthermore, inthis variation, Block S250 can additionally comprise depositing a fillermaterial within regions between the electronics substrate and theheater-sensor die not connected by a volume of solder, in order tofurther stabilize the assembly. In other variations, the coupling canadditionally or alternatively comprise any suitable adhesive (e.g.,cyanoacrylate adhesive).

Wire bonding in variations of Block S250 can comprise any suitablecombination of the above variations, and can additionally oralternatively comprise any suitable encapsulation, embedding, or pottingof wire bonds to further prevent failure in the wire bonds. Furthermore,while variations of Block S250 are described for coupling of a set ofheater-sensor dies to an electronics substrate, Block S250 canalternatively comprise coupling of a single heater-sensor die to theelectronics substrate, in order to produce a single heating surfaceconfigured to heat a detection chamber in thermal communication with theheater-sensor die. However, Block S250 can alternatively comprisecoupling any suitable number of heater-sensor dies to any suitablenumber of electronics substrates.

As shown in FIG. 10, the method 200 can further comprise Block S260,which recites forming a second insulating layer upon the heating region.Block S260 functions to electrically isolate the heating region on afirst side and a second side of the heating region, and is preferablyperformed prior to coupling of the set of heater-sensor dies to theelectronics substrate. However, Block S260 can alternatively beperformed at any other suitable time relative to other blocks of themethod 200. Preferably, Block S260 comprises depositing (e.g.,electrodepositing, using CVD) or growing (e.g., by thermal oxidation) anoxide on the heating region, such that the heating region is“sandwiched” between two oxide layers; however, Block S260 canadditionally or alternatively comprise depositing any other suitableinsulating material by any suitable method at another surface of theheating region. In one variation, Block S260 comprises depositing a lowtemperature oxide by chemical vapor deposition (e.g., plasma-enhancedchemical vapor deposition) to form the second insulating layer, and inother variations, Block S260 can comprise fluid deposition of aninsulating material (e.g., inkjet printing or casting of an electricallyinsulating polymer, softbaking/hardbaking of a deposited polymer, etc.)onto desired portions of the heating region.

In variations of the method 200 comprising Block S260, the method 200can also further comprise Block S265, which recites removing material ofthe second insulating layer to produce a set of connection points. BlockS265 functions to provide access to the heating region betweeninsulation regions, such that the heating region can be electricallyconnected to the electronics substrate. The set of connection points canbe defined using a material removal method including any one or more of:etching (e.g., lithography, laser etching), machining (e.g., drilling),and any other suitable method of material removal. In one such variationof Block S265, the set of connection points can be definedphotolithographically using a positive resist process in a mannersimilar to that used in a variation of Block S230. In one example ofthis variation, the second insulating layer can be covered with positivephotoresist and lithographically etched in exposed regions. In theexample, the positive photoresist can then be removed to reveal theconnection points. In other variations, the connection points can bedefined using any lithographic method, using positive and/or negativeetching to form the heating pattern, and/or using any other suitablemethod. Upon definition of the connection points, the second insulatinglayer can further be etched (e.g., using buffered hydrofluoric acid) asan additional surface treatment. Alternative variations of Block S265can include additionally or alternatively removing material from thefirst insulating surface of Block S210 to form any subset of the set ofconnection points.

As shown in FIG. 10, the method 200 can further include Block S270,which recites: coupling a non-reflective coating to at least oneheater-sensor die of the set of heater-sensor dies. Block S270 functionsto process at least a subset of the set of heater-sensor dies 110 sothat they do not interfere with light transmitted to photodetectors ofan optical subsystem opposed (e.g., directly opposed, in opposition) tothe set of heater-sensor dies no (e.g., light emitted from the set ofbiological samples, light transmitted through filters of an opticalsubsystem), especially in configurations wherein a set of detectionchambers is configured between the set of heater-sensor dies and opticalelements of an optical subsystem. The non-reflective coating ispreferably coupled identically to all heater-sensor dies of the set ofheater sensor dies; however, the non-reflective coating canalternatively be coupled non-identically to one or more heater-sensordies of the set of heater-sensor dies. As such, in variations, one ormore subsets of the set of heater-sensor dies can be coupled tonon-reflective coatings in a manner that provides different lightreflection properties for the subset(s) of the set of heater-sensordies.

In Block S270, the non-reflective coating is preferably a material layerthat is applied superficial to at least one of the first insulatinglayer and the second insulating layer processed in variations of BlocksS210 and S260, respectively. In one example, the non-reflective coatingprocessed in Block S270 can comprise a high-temperature paint (e.g.,dark paint, flat paint) that functions to absorb and/or diffuse lightfrom an opposing optical subsystem, while facilitating heat transfer toa set of detection chambers in thermal communication with the set ofheater-sensor dies. In this example, the high-temperature paint can beapplied by any one or more of: brushing, spraying, dipping, printing,and any other suitable method of coupling the high-temperature paint toone or more surfaces of at least a subset of the set of heater-sensordies. However, the non-reflective coating can alternatively be processedsimultaneously with or can comprise one or more of the first insulatinglayer and the second insulating layer processed in variations of BlocksS210 and S260, respectively. For instance, one or more of the first andthe second insulating layer can include a modified oxide layer that haslow-reflectivity, thus preventing interference caused by light reflectedfrom the set of heater-sensor dies. In some extreme variations, however,mitigation of interference due to reflected light from the set ofheater-sensor dies can be produced by configuring the set ofheater-sensor dies to be in misalignment with photodetectors of theoptical subsystem, such that reflection does not interfere with lighttransmitted to the photodetectors of the optical subsystem, in modifiedversions of Block S270. For instance, the set of heater-sensor dies canbe configured to heat a set of detection chambers from a firstdirection, and the optical subsystem can be configured to receive lightfrom the set of detection chambers from a second direction (e.g., adirection non-parallel to the first direction), such that reflectionfrom the set of heater-sensor dies 110 does not cause interference. Instill other variations of Block S270, the set of heater-sensor dies canbe processed with any other suitable elements (e.g., coatings, layers,etc.) and/or be configured in any other suitable manner that eliminates,prevents, or mitigates reflection from the set of heater-sensor diesfrom interfering with light transmitted to photodetectors of an opticalsubsystem in opposition to the set of heater-sensor dies no.

The method 200 can further comprise any other suitable block, such ascalibrating sensing elements of the thermocycling system S280. In anexample of Block S270, the set of heater-sensor dies coupled to theelectronics substrate can be installed in thermal chamber to calibratethe sensing elements (i.e., RTDs) of the set of heater-sensor dies. Inthe example, the electronics substrate and a first connector end of acalibration system was placed in a thermal chamber and a secondconnector end of the calibration system was attached to an array ofcontacts outside the thermal chamber. The thermal chamber was allowed toequilibrate in stages at a series of temperatures spanning the expecteddynamic range of the RTDs, from 30 C to 100 C in four stages. The RTDresistance values were read out at the various equilibratedtemperatures, and fit a Callendar-Van Dusen equation. The calibration ofthe example of Block S270 yielded the coefficients used to convert thesensing element resistance values to temperature values, in order tocalibrate the sensing elements of the system.

The system 100 and/or method 200 of the preferred embodiment andvariations thereof can be embodied and/or implemented at least in partas a machine configured to receive a computer-readable medium storingcomputer-readable instructions. The instructions are preferably executedby computer-executable components preferably integrated with the system300 and one or more portions of the processor 350. The computer-readablemedium can be stored on any suitable computer-readable media such asRAMs, ROMs, flash memory, EEPROMs, optical devices (CD or DVD), harddrives, floppy drives, or any suitable device. The computer-executablecomponent is preferably a general or application specific processor, butany suitable dedicated hardware or hardware/firmware combination devicecan alternatively or additionally execute the instructions.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of methods according to preferred embodiments,example configurations, and variations thereof. In this regard, eachblock in the flowchart or block diagrams may represent a module,segment, or portion of code, which comprises one or more executableinstructions for implementing the specified logical function(s). Itshould also be noted that, in some alternative implementations, thefunctions noted in the block can occur out of the order noted in theFIGURES. For example, two blocks shown in succession may, in fact, beexecuted substantially concurrently, or the blocks may sometimes beexecuted in the reverse order, depending upon the functionalityinvolved. It will also be noted that each block of the block diagramsand/or flowchart illustration, and combinations of blocks in the blockdiagrams and/or flowchart illustration, can be implemented by specialpurpose hardware-based systems that perform the specified functions oracts, or combinations of special purpose hardware and computerinstructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A system for thermocycling biological samples within detection chambers comprising: a set of heater-sensor dies, each heater-sensor die 111 the set of heater-sensor dies comprising a heating surface configured to interface with a detection chamber and a second surface, inferior to the heating surface, including a first connection point; an electronics substrate, comprising a first substrate surface coupled to the second surface of each of the set of heater-sensor dies, an aperture providing access through the electronics substrate to at least one of the set of heater-sensor dies, and a second substrate surface inferior to the first substrate surface, wherein the electronics substrate comprises a set of substrate connection points at least at one of the first substrate surface, an aperture surface defined within the aperture, and the second substrate surface, and wherein the electronics substrate is configured to couple heating elements and sensing elements of the set of heater-sensor dies to a controller; and a set of wire bonds, including a wire bond coupled between the first connection point of at least one of the set of heater-sensor dies and one of the set of substrate connection points.
 2. The system of claim 1, wherein the electronics substrate includes a set of apertures longitudinally spaced across the electronics substrate, and wherein the first connection point of each of the set of heater-sensor dies is accessible through at least one aperture of the set of apertures.
 3. The system of claim 2, wherein the set of substrate connection points is distributed at regions of the second substrate surface between adjacent apertures of the set of apertures of the electronics substrate.
 4. The system of claim 2, wherein the electronics substrate further includes a set of recesses, each recess of the set of recesses contiguous with at least one aperture of the set of apertures.
 5. The system of claim 4, wherein each recess of the set of recesses is configured to receive a heater-sensor die of the set of heater-sensor dies.
 6. The system of claim 4, wherein each heater sensor-die of the set of heater-sensor dies is coupled to the first substrate surface, and the set of substrate connection points is defined between the first substrate surface and the second substrate surface within the set of recesses.
 7. The system of claim 2, further including a set of heat-sink supports coupled to at least one of the set of heater-sensor dies, through the set of apertures, and the second substrate surface of the electronics substrate and configured to dissipate heat generated by the set of heater-sensor dies.
 8. The system of claim 7, wherein at least one of the set of heat-sink supports includes an integrated cooling element of a cooling subsystem, and wherein a base surface of each of the set of heat-sink supports is coupled to an elastic element configured for transmit a biasing force through the electronics substrate, thereby maintaining thermal communication between the set of heater-sensor dies and a set of detection chambers upon alignment of the set of heater-sensor dies with the set of detection chambers.
 9. The system of claim 1, wherein the electronics substrate is a flexible electronics substrate, and wherein the system further comprises a set of elastic elements coupled to the second substrate surface of the flexible electronics substrate and configured to transmit a set of biasing forces through the flexible electronics substrate and to the set of heater-sensor dies, thereby maintaining thermal communication between the set of heater-sensor dies and a set of detection chambers upon alignment of the set of heater-sensor dies with the set of detection chambers.
 10. The system of claim 1, wherein each of the set of heater-sensor dies includes a coating, proximal the heating surface, configured to mitigate reflection of light from the heating surface toward photodetectors of an optical subsystem, in a configuration wherein the set of heater-sensor dies is opposed to photodetectors of the optical subsystem.
 11. A method of manufacturing a system for thermocycling biological samples within detection chambers, the method comprising: at a substrate, forming a first insulating layer coupled to exposed surfaces of the substrate; depositing a heating region onto the first insulating layer of the substrate; removing material of the heating region, thereby forming a heating array with a pattern that defines a set of heating elements and a set of sensing elements within the heating array; dividing the heating array into a set of heater-sensor dies, each heater-sensor die comprising a heating surface, including a heating element of the set of heating elements and a sensing element of the set of sensing elements, configured to interface with a detection chamber, and each heater-sensor die comprising a second surface, inferior to the heating surface, including a first connection point; providing an electronics substrate having a first substrate surface configured to couple to the second surface of each of the set of heater-sensor dies, an aperture providing access through the electronics substrate to at least one of the set of heater-sensor dies, and a second substrate surface inferior to the first substrate surface; and coupling a wire bond from the first connection point of at least one of the set of heater-sensor dies, into the aperture of the electronics substrate, and to a second connection point of the electronics substrate.
 12. The method of claim 11, wherein depositing the heating region comprises coupling an adhesion material layer to the first insulating layer, and coupling a noble material layer to the adhesion material layer.
 13. The method of claim 12, wherein forming the heating array with a pattern comprises defining a pattern of voids through the adhesion material layer and the noble material layer, wherein coarse elements of the pattern define heating elements of the heating array and fine elements of the pattern, integrated within coarse elements of the pattern, define sensing elements of the heating array.
 14. The method of claim 13, wherein the pattern of the heating array comprises boustrophedonic segments including wide segments associated with heating elements of the heating array and fine segments, surrounded by wide segments, associated with sensing elements of the heating array.
 15. The method of claim 11, wherein coupling the wire bond comprises coupling a first end of the wire bond to the first connection point, passing the wire bond through the aperture, and coupling a second end of the wire bond to the second connection point at the second substrate surface.
 16. The method of claim 11, wherein coupling the wire bond comprises coupling a first end of the wire bond to the first connection point, passing the wire bond into the aperture, and coupling a second end of the wire bond to a surface of the aperture within the electronics substrate.
 17. The method of claim 11, wherein providing the electronics substrate comprises defining a set of apertures longitudinally spaced across the electronics substrate and a set of recesses, each recess of the set of recesses contiguous with at least one aperture of the set of apertures.
 18. The method of claim 17, further comprising coupling the second surface of each of the set of heater-sensor dies, within a recess of the set of recesses, with an adhesive layer configured to maintain coupling between the set of heater-sensor dies and the electronics substrate.
 19. The method of claim 17, wherein coupling the wire bond comprises coupling a heater-sensor dies of the set of heater-sensor dies to the first substrate surface with an adhesive layer, and coupling a first end of the wire bond to the first connection point, passing the wire bond into a recess of the set of recesses, and coupling a second end of the wire bond to the second connection point at a surface of at least one of the recess and an aperture contiguous with the recess.
 20. The method of claim 11, further comprising coupling a coating, proximal to the heating surface of at least one heater-sensor die of the set of heater-sensor dies, wherein the coating is configured to mitigate reflection of light from the heating surface toward photodetectors of an optical subsystem, in a configuration wherein the set of heater-sensor dies is opposed to photodetectors of the optical subsystem. 